Aging causes thymic involution which decreases thymic lymphopoiesis and exhausts the naive T-cell pool, constricting diversity of T-cell receptor repertoire and inducing immunosenescence. However, the mechanisms related to cellular compartments underlying thymic involution are unclear. The two main cellular compartments in the thymus are lymphohematopoietic progenitor cells (LPCs) and thymic epithelial cells (TECs). Currently, it is controversial if LPCs develop a cumulative intrinsic defect with age to trigger thymic involution, or whether aging results in dysfunction of TECs, causing secondary changes in thymocytes and thymic involution. Based on our preliminary studies, we hypothesize that the primary/dominant defect in aging is dysfunction of TECs, which in turn causes age-related thymopoietic insufficiency, in part through inadequate Notch gene signals that affect early stages of T-cell development. We will test these hypotheses through the following specific aims: 1). Compare the capacity of LPCs from middle-aged/aged and young animals to competitively develop in thymic stromal niches of young animals. We will measure competitive repopulation of unirradiated young IL-7R-/- recipient thymi by LPCs from old and young mice, to determine if the former have any intrinsic and irreversible defects. We will use a second competitive model, transplanting a fetal TEC network from RAG-/- mice to the kidney capsule of young RAG-/- mice, followed by intravenous administration of LPCs from old and young mice. 2). To determine whether the thymic microenvironment in aging provides inadequate Notch signals, resulting in reduced thymic lymphopoiesis. We will analyze expression of Notch ligands in TECs, and Notch receptors and Notch target genes in early stage thymocytes from aged mice. Then, we will provide enhanced Notch signaling to aged thymus in vivo by infusing Notch ligand-expressing thymic epithelial cell lines. We will then determine whether the enhanced Notch signaling can improve early stages of T-cell development in the aged thymus. The proposed studies will improve our understanding of the mechanism(s) of aging-related decreased T-lymphopoiesis and lay the groundwork for development of practical strategies to combat thymopoietic failure due to aging. PUBLIC HEALTH RELEVANCE: This proposal will identify the cellular compartment which has the dominant/primary defect that causes aging-related thymic involution, and determine whether inadequate Notch signals contribute to this defect by impairing early T-cell development and T-lymphopoiesis in the elderly.